The role of Ca++, calmodulin and hydrophobic interactions in the activation of Bordetella pertussis adenylate cyclase has been extended. A stable spheroplast membrane preparation has been developed for a very sensitive assay procedure to as little as less than 1 femtomole of the regulator protein. Hydrophobic interactions are shown by 3-10-fold stimulation by various amphiphiles that is not dependent on micelle formation but does depend on Ca++. Nevertheless, we have now found conditions where we can demonstrate a Ca++ independent activation of the B. pertussis adenylate cyclase by calmodulin and the basis of this interaction is under study.